Fanconi anemia (FA) is a recessively inherited disease characterized by congenital defects, bone marrow failure, and cancer susceptibility. The disease has also been considered by some as a segmental progeriod entity, as FA patients develop a range of tissue-specific premature onset and accelerated aging phenotypes, including bilateral cataracts, skin atrophy, reduced muscle mass, premature ovarian failure, higher frequency of osteoporosis and osteopenia, and diabetes. Sixteen genes have now been described that are mutated to cause FA. Three of them are discovered by our group. Recent evidence suggests that FA proteins function in a DNA damage response pathway involving the proteins produced by the breast cancer susceptibility genes BRCA1 and BRCA2. A key step in this pathway is modification of two FA proteins, FANCD2 and FANCI. The modification, monoubiquitylation, results in redistribution of FANCD2-FANCI to specific spots in the nucleus where BRCA proteins also localize. When we initiated this project in 2001, five FA proteins (FANCA, -C, -E, -F, and -G) were found to interact with each other to form a multiprotein nuclear complex, the FA core complex. This complex functions upstream in the pathway and is required for FANCD2-FANCI monoubiquitylation. We purified the FA core complex and found that it contains at least eight new components in addition to the five known FA proteins. We have characterized these new components and shown that they are important for the FA-associated DNA damage response pathway, as summarized below. One new component, termed FANCL, possesses ubiquitin ligase activity in vitro and is essential for FANCD2-FANCI monoubiquitylation in vivo. FANCL is defective in a group of Fanconi anemia patients, and therefore represents a novel Fanconi anemia gene. FANCL plays a crucial role in the Fanconi anemia pathway as the catalytic subunit for monoubiquitylation and FANCD2-FANCI. FANCL might be a potential target for new therapeutic intervention. The 95 kd subunit of the Fanconi anemia core complex is defective in FA complementation group B patients (the gene is named FANCB). FANCB is X-linked and present in only one active copy in normal cells. Thus, FANCB could represent a vulnerable target in the machinery that maintains genome stability. The 250 Kda subunit of the FA core complex, named FANCM, is mutated in FA patients of a new complementation group, FA-M. FANCM has a conserved helicase domain and a DNA remodeling activity. FANCM has at least three important roles in the FA DNA damage response pathway. First, it plays a structural role, allowing assembly of the FA core complex, because in its absence, the nuclear localization and stability of several FA proteins are defective. Second, FANCM translocates and remodels various DNA structures, which are important for subsequent DNA repair. Third, FANCM is hyperphosphorylated in response to DNA damage, suggesting that it may serve as a signal transducer through which the activity of the core complex is regulated. We identified the 100 Kda subunit of the FA core complex, FAAP100, and our collaboration with another group shows that it is also defective in an FA patient and required for FANCD2 monoubiquitination. We identified the 24 Kda subunit of the FA core complex, termed FAAP24, which forms a heterodimer with FANCM. FAAP24 can recognize structured DNA that mimics intermediates generated during DNA replication. Moreover, it can target FANCM to such structures. Cells depleted of FAAP24 show phenotypes that are characteristics of FA cells. We collaborated with other labs to demonstrate that PALB2, a partner of BRCA2, is the gene defective in Fanconi anemia complementation group N patients. In mechanistic studies, we demonstrated that FANCM possesses an ATP-independent binding activity and an ATP-dependent bi-directional branch-point translocation activity on a synthetic four-way junction DNA, which mimiics intermediates generated during homologous recombination or at stalled replication forks. We found that the ATP-dependent activities of FANCM are required for cellular resistance to a DNA crosslinking drug, mitomycin C (MMC), but not for the monoubiquitination of FANCD2-FANCI. In contrast, monoubiquitination requires the entire helicase domain of FANCM, which has both ATP- dependent and independent activities. These data are consistent with participation of FANCM and its associated FA core complex in the FA pathway at both signaling through monoubiquitination and the ensuing DNA repair. We identified two new components in the FA core complex, MHF1 and MHF2. These two proteins form a histone-fold heterodimer that associates with FANCM to form a DNA-remodeling complex conserved from yeast to human. MHF stimulates DNA binding and replication fork remodeling by FANCM. In the cell, FANCM and MHF are rapidly recruited to forks stalled by DNA interstrand crosslinks, and both are required for cellular resistance to such lesions. Notably, the yeast orthologs of these proteins also function together to resist MMS-induced DNA damage and promote gene conversion at blocked replication forks. Thus, FANCM-MHF is an essential DNA-remodeling complex that protects replication forks from yeast to human. We showed that another FA protein, FANCJ, becomes hyperphosphorylated in response to DNA damage. We are investigating if this phosphorylation regulates activity of FANCJ in DNA repair. We collaborated with Dr. Lei Li's lab to develop a chromatin-IP-based strategy termed eChIP and elucidated how various FA proteins are recruited to the interstrand DNA crosslinks that block replication. We found that BRCA-related FA proteins (BRCA2, FANCJ/BACH1, and FANCN/PALB2), but not FA core and I/D2 complexes, require replication for their crosslink association. FANCD2, but not FANCJ and FANCN, requires the FA core complex for its recruitment. FA core complex requires nucleotide excision repair proteins XPA and XPC for its association. Thus, FA proteins participate in distinct DNA damage response mechanisms governed by DNA replication status. We found that the FA network is regulated by a ubiquitin signaling cascade initiated by RNF8 and its partner, UBC13, and mediated by FAAP20, a new component of the FA core complex. FAAP20 preferentially binds the ubiquitin product of RNF8-UBC13, and this ubiquitin-binding activity and RNF8-UBC13 are both required for recruitment of FAAP20 to ICLs. Both RNF8 and FAAP20 are required for recruitment of FA core complex and FANCD2 to ICLs, whereas RNF168 can modulate efficiency of the recruitment. RNF8 and FAAP20 are needed for efficient FANCD2 monoubiquitination, a key step of the FA network; RNF8 and the FA core complex work in the same pathway to promote cellular resistance to ICLs. Thus, the RNF8-FAAP20 ubiquitin cascade is critical for recruiting FA core complex to ICLs and for normal function of the FA network. We have worked with Lei Li's lab and showed that FANCM and FAAP24 can work either cooperatively or in parallel pathways to activate the FA network and protect genome stability. We have worked with Michael Seidman's group to show that DNA ICLs are not absolute blocks for replication, and one function of FANCM-MHF complex is to promote replication machinery to traverse the ICLs. Moreover, we found that the BLM complex is also required for this traverse activity. More recently, we and our collaborators found that FANCM can directly interact with replication machinery to promote the traverse reaction. We have worked with Wei Yang's lab and solved the crystal structure of MHF with or without its interaction domain of FANCM. We found that FANCM remodels the structure of MHF to recognize branched DNA, such as replication forks or Holliday junctions, and to protect genome stability.